Application of dWIZ-1 and salt thereof in preparation of tumor immunotherapy synergist

Abstract

The invention belongs to the field of medicines, and discloses application of dWIZ-1 and salt thereof in preparation of a tumor immunotherapy synergist. The inventor finds that the dWIZ-1 or PD-1 monoclonal antibody does not obviously inhibit the growth of tumors accompanied by chronic inflammation when being independently used, but the dWIZ-1 and the PD-1 monoclonal antibody can obviously reduce immunosuppressive cells in the tumors, obviously increase immune effector cells (CD8 + T cells) and obviously inhibit the growth of the tumors by combining the dWIZ-1 and the PD-1 monoclonal antibody.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of dWIZ-1 and its salts in the preparation of an enhancer for tumor immunotherapy. Background Art

[0002] In recent years, immunotherapy drugs represented by PD-1 monoclonal antibodies have shown great potential in the treatment of tumors and have attracted increasing attention. The advantages of immunotherapy include high specificity, durable efficacy, broad spectrum, potential for combination therapy, and providing hope for advanced patients. The disadvantages include limited efficiency, severe side effects, high cost, drug resistance, imperfect biomarkers, insufficient long-term data, large individual differences, limited effects on solid tumors, and complex preparation. The effect of tumor immunotherapy is affected by multiple factors. Using enhancers to improve the efficacy of tumor immunotherapy has very practical significance. Since most liver cancers are combined with chronic liver inflammation, their immunosuppressive environment leads to worse treatment effects and lower efficiency of PD-1 monoclonal antibodies compared with other solid tumors. Therefore, developing new drug treatment strategies to improve the efficiency of PD-1 monoclonal antibodies is of great significance for the treatment of liver cancer under the background of chronic inflammation.

[0003] WIZ (Widely Interspaced Zinc Finger Motif Protein), as a transcription factor, is widely involved in gene expression regulation, chromatin state maintenance, and interaction with other transcription complexes. Although the development of WIZ inhibitors is difficult, as a potential "underexplored" target, it has extremely high research and treatment potential. dWIZ-1 ( ), and dWIZ-2 ( ) are two small molecule drugs developed by Novartis. They belong to a class of innovative drugs called "molecular glue degraders". These drugs hijack the intracellular protein degradation mechanism to selectively degrade pathogenic proteins, thereby achieving the purpose of treating diseases. These two molecular glues bind to WIZ and the E3 ubiquitin ligase CRBN, inducing the degradation of the WIZ protein, thus overcoming the limitation that traditional small molecule drugs are difficult to target transcription factors. Research shows that reducing the level of the WIZ protein can upregulate the expression of fetal hemoglobin (HbF), thereby improving the symptoms of sickle cell disease. Therefore, dWIZ-1 and dWIZ-2 are expected to become new therapies for the treatment of sickle cell disease. There is no research indicating the correlation between dWIZ-1 and immunotherapy. Summary of the Invention

[0004] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide the use of dWIZ-1 and its salts in the preparation of an enhancer for tumor immunotherapy.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In the first aspect of the present invention, there is provided:

[0007] The use of dWIZ-1 and its salts in the preparation of a tumor immunotherapy synergist.

[0008] In some application examples, the tumor is liver cancer.

[0009] In some application examples, the liver cancer is accompanied by inflammation.

[0010] In some application examples, the liver cancer is accompanied by chronic liver inflammation.

[0011] In some application examples, the tumor immunotherapy is PD-1 monoclonal antibody or PD-L1 monoclonal antibody immunotherapy.

[0012] In some application examples, the PD-1 monoclonal antibody is selected from tislelizumab, camrelizumab, nivolumab, pembrolizumab, and sintilimab.

[0013] In some application examples, the synergist is an injection.

[0014] In some application examples, the synergist further includes excipients.

[0015] In the second aspect of the present invention, there is provided:

[0016] A composition comprising a PD-1 monoclonal antibody / PD-L1 monoclonal antibody having a therapeutic effect on tumors and a PD-1 monoclonal antibody / PD-L1 monoclonal antibody immunotherapy synergist, wherein the synergist is dWIZ-1 or its salt.

[0017] In some composition examples, the PD-1 monoclonal antibody is selected from tislelizumab, camrelizumab, nivolumab, pembrolizumab, and sintilimab.

[0018] In some composition examples, the tumor is liver cancer.

[0019] In some composition examples, the liver cancer is accompanied by inflammation.

[0020] In some composition examples, the liver cancer is accompanied by chronic liver inflammation.

[0021] The beneficial effects of the present invention are as follows:

[0022] The inventors have found through research that the use of dWIZ-1 or PD-1 monoclonal antibody alone has no obvious inhibitory effect on the growth of tumors accompanied by inflammation, while the combination of dWIZ-1 and PD-1 monoclonal antibody can significantly reduce the immunosuppressive cells in the tumor and the immune effector cells (CD8+ The (T cells) increased significantly, significantly inhibiting tumor growth. Description of the Drawings

[0023] Figure 1 It is the experimental result of the effect of dWIZ-1 and dWIZ-2 on the growth of liver cancer in mice with chronic liver inflammation, showing that it cannot significantly inhibit the tumor growth of mice (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant).

[0024] Figure 2 It is the experimental result that dWIZ-1 and the PD-1 monoclonal antibody (tislelizumab) alone have no obvious inhibitory effect on the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant), while the combination of dWIZ-1 and the PD-1 monoclonal antibody (tislelizumab) can significantly inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant).

[0025] Figure 3 It is the experimental result that dWIZ-2 and the PD-1 monoclonal antibody (tislelizumab) alone have no obvious inhibitory effect on the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant), and the combination of dWIZ-2 and the PD-1 monoclonal antibody (tislelizumab) cannot inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant).

[0026] Figure 4 It is that the PD-1 monoclonal antibody (tislelizumab) has no obvious effect on immunosuppressive cells (MDSCs) and immune effector cells (CD8 + T cells) in the liver cancer of mice with chronic liver inflammation ( Figure 4 A), but after treatment with dWIZ-1 alone or in combination with the PD-1 monoclonal antibody (tislelizumab), the immunosuppressive cells (MDSCs) in the liver cancer of mice are significantly reduced, and the immune effector cells (CD8 + T cells) increase significantly ( Figure 4 B) experimental results. Detailed Implementation Manner

[0027] The technical solution of the present invention will be further described below in combination with experiments.

[0028] Experimental Method:

[0029] NSI mice are severely immunodeficient mice lacking immune cells such as B, T, and NK cells. In our previous study, we constructed immune humanized NSI mice using PBMCs, which can accurately reflect the efficacy of anti-PD-1 monoclonal antibody against tumors (Shouheng Lin, Guohua Huang, et al. Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1 / PD-1 targeted immunotherapy [J]. MAbs, 2018, 10:1301-1311). The modeling is as follows: Collect peripheral blood; obtain PBMCs through steps such as diluting the blood, adding lymph node separation solution, centrifuging (2000 rpm, 20 minutes), aspirating the cells and resuspending them; 4-week-old NSI mice receive 0.5 Gy of sublethal irradiation, and 5×10 6 PBMCs are injected into the body through the tail vein 4 hours later.

[0030] The chronic inflammatory environment of the liver caused by different etiologies has the same pathological process: pathogenic factors such as viruses and alcohol continuously damage hepatocytes, and the latter induce and maintain the liver inflammatory state through pathways such as releasing cytokines and chemokines, activating Kupffer cells and hepatic stellate cells, and recruiting peripheral inflammatory cells. Mice can be induced to have chronic liver inflammation by intragastric administration of CCl 4 , which can better simulate the pathological characteristics of the liver in clinical liver cancer patients (Methods Mol Biol 2017, 1559: 279-296; Front. Physiol 2021, 12:666138). Therefore, we plan to construct relevant models based on immune humanized NSI mice. The steps are briefly described as follows: After injecting PBMCs, NSI mice are given 40% CCl 4 by intragastric administration to induce chronic liver inflammation in mice (0.1 ml, 3 times a week) for 4 weeks; Take the well-growing human hepatocellular carcinoma cell line HepG2, and configure the cells into a suspension (4×10 7 / ml) using PBS and Matrigel (BD Biocoat, Cat. NO. 354262) in an equal volume ratio; Anesthetize the mice by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), make a mid-upper abdominal incision and expose the left lateral lobe of the liver, and inject the cell suspension containing HepG2 into the middle and outer 1 / 3 of the left lateral lobe of the mouse liver (the injection volume is 25 μl / mouse, and the cell number is 1×10 6( / mouse); Gently press the injection site with a cotton swab to prevent bleeding and extracellular leakage. After confirming successful injection, suture the incision layer by layer. Through the above operations, a mouse orthotopic transplantation tumor model of liver cancer with chronic liver inflammation can be obtained. In addition, replace CCl in the first step of the above model construction with its solvent olive oil for gavage, and the subsequent operation steps are the same, then a mouse orthotopic transplantation tumor model of liver cancer with normal liver can be obtained. The mouse models we constructed have been successfully verified (Yuchuan Jiang, Siliang Chen, et al. TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration, Front Immunol, 2021; 12: 612139). 4 Change it to its solvent olive oil for gavage, and the subsequent operation steps are the same, then a mouse orthotopic transplantation tumor model of liver cancer with normal liver can be obtained. The mouse models we constructed have been successfully verified (Yuchuan Jiang, Siliang Chen, et al. TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration, Front Immunol, 2021; 12:612139).

[0031] Divide the immune humanized mouse orthotopic transplantation tumor model of liver cancer with chronic liver inflammation into a control group, a dWIZ-1 treatment group, a dWIZ-2 treatment group, and a combined dWIZ-1 and dWIZ-2 treatment group. dWIZ-1 and dWIZ-2 start gavage administration 3 days after the construction of the mouse model, with a single dose of 10 mg / kg; the control group is given the same dose of normal saline by gavage. After 7 days, the mice are anesthetized and euthanized, and the tumors are removed to evaluate the volume of the primary focus and the liver weight / body weight ratio ( Figure 1 ).

[0032] Divide the immune humanized mouse orthotopic transplantation tumor model of liver cancer with chronic liver inflammation into a control group, a dWIZ-1 treatment group, a PD-1 monoclonal antibody treatment group, and a combined dWIZ-1 and PD-1 monoclonal antibody treatment group. The drug used in the PD-1 monoclonal antibody treatment group is tislelizumab injection, which is purchased (National Medicine Approval Number S20190045) from BeiGene. Tail vein injection administration starts 3 days after the construction of the mouse model, with a single dose of 2 mg / kg; the control group is given the same dose of normal saline by tail vein injection, and the administration of dWIZ-1 is the same as before. After 7 days, the mice are anesthetized and euthanized, and the tumors are removed to evaluate the volume of the primary focus and the tumor weight / body weight ratio ( Figure 2 ).

[0033] The orthotopic transplantation tumor model of humanized immune mouse liver cancer with chronic liver inflammation was divided into a control group, a dWIZ-2 treatment group, a PD-1 monoclonal antibody treatment group, and a combined dWIZ-2 and PD-1 monoclonal antibody treatment group. The drug used in the PD-1 monoclonal antibody treatment group was tislelizumab injection, which was purchased (National Medicine Approval Number S20190045) from BeiGene. After the mouse model was constructed, tail vein injection was started on the 3rd day, with a single dose of 2 mg / kg. The control group was given the same dose of normal saline by tail vein injection. The administration of dWIZ-2 was the same as before. After 7 days, the mice were anesthetized and euthanized, and the tumors were removed to evaluate the volume of the primary focus and the ratio of tumor weight to body weight ( Figure 3 ).

[0034] Detection Figure 2 of MDSCs and CD8 + T cells ( Figure 4 ) in the liver cancer tissues of the 4 groups of mice: The isolation of high-purity primary tumor cells was mainly achieved through steps such as tumor perfusion digestion, tumor ex vivo digestion, and high-speed centrifugation. Prepare the perfusion device and anesthetize the mice; place the tumor into the indwelling needle, connect the perfusion device and run it, and stop perfusion when the tumor turns pure white and cracks on the surface; separate the tumor and transfer it into a culture dish containing digestive fluid (protease / collagenase with 1% Dnase I), filter it through a 40 μm cell sieve after sufficient digestion to obtain a single-cell suspension; centrifuge the cell suspension at 51 g and 4°C for 5 minutes, add 10 ml of perfusion fluid to resuspend the obtained precipitate, centrifuge again under the same conditions, and remove the obtained precipitate; place it into a Ficoll lymphocyte separation solution, add an appropriate amount of 1640 medium to keep the separation boundary distinct, centrifuge at 800 g at room temperature for 30 minutes, aspirate the stratified cells after gradient centrifugation, add 1640 medium to resuspend and wash, add FCR antibody blocker, add FCR antibody blocker, and divide the cell suspension into two parts. One part is stained with fluorescent-labeled CD45, CD11b, Ly6G, and Ly6C flow antibodies, and the other part is stained with CD45, CD3, and CD8 flow antibodies to detect the number of tumor MDSCs and the infiltration of CD8 + T cells ( Figure 4 ).

[0035] The experimental results are as Figures 1 to 4 shown.

[0036] Figure 1 The treatment results of dWIZ-1 and dWIZ-2 on the growth of liver cancer in mice with chronic liver inflammation were shown, indicating that they could not significantly inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in the tumor volume and liver weight / body weight ratio between the treatment group and the control group were not significant).

[0037] Figure 2It is indicated that the use of dWIZ-1 and anti-PD-1 monoclonal antibody (tislelizumab) alone does not significantly inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant), while the combination of dWIZ-1 and anti-PD-1 monoclonal antibody (tislelizumab) can significantly inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant).

[0038] Figure 3 It is indicated that the use of dWIZ-2 and anti-PD-1 monoclonal antibody (tislelizumab) alone does not significantly inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant), and the combination of dWIZ-2 and anti-PD-1 monoclonal antibody (tislelizumab) also cannot inhibit the growth of liver cancer in mice with chronic liver inflammation (the differences in tumor volume and liver weight / body weight ratio between the treatment group and the control group are not significant).

[0039] Figure 4 It is indicated that anti-PD-1 monoclonal antibody (tislelizumab) has no obvious effect on immunosuppressive cells (MDSCs) and immune effector cells (CD8 + T cells) in the liver cancer of mice with chronic liver inflammation ( Figure 4 A), but after treatment with dWIZ-1 alone or in combination with anti-PD-1 monoclonal antibody (tislelizumab), the immunosuppressive cells (MDSCs) in the liver cancer of mice with chronic liver inflammation are significantly reduced, and the immune effector cells (CD8 + T cells) are significantly increased ( Figure 4 B).

[0040] The experimental data also show that other anti-PD-1 monoclonal antibodies, such as camrelizumab, nivolumab, pembrolizumab and sintilimab, also have a synergistic effect when combined with dWIZ-1, and the immunotherapeutic efficacy of the anti-PD-1 monoclonal antibody is further improved after combination.

[0041] The above is a further detailed description of the present invention, and it should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention belongs, any simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. Application of dWIZ-1 and its salts in the preparation of tumor immunotherapy enhancers.

2. The use according to claim 1, characterized in that: The tumor is liver cancer.

3. The use according to claim 2, characterized in that: The liver cancer is accompanied by inflammation.

4. The use according to claim 3, characterized in that: The liver cancer is accompanied by chronic liver inflammation.

5. The use according to any one of claims 1 to 4, characterized in that: The tumor immunotherapy is PD-1 monoclonal antibody or PD-L1 monoclonal antibody immunotherapy.

6. The use according to claim 5, characterized in that: The PD-1 monoclonal antibody is selected from tilelizumab, carrelizumab, nivolumab, pembrolizumab and sintilimab.

7. The use according to any one of claims 1 to 4, characterized in that: The synergist is an injection.

8. The use according to any one of claims 1 to 4, characterized in that: The synergist also includes auxiliary materials.

9. A composition comprising a PD-1 monoclonal antibody / PD-L1 monoclonal antibody having a therapeutic effect on tumors and a PD-1 monoclonal antibody / PD-L1 monoclonal antibody immunotherapy enhancer, characterized in that: The synergist is dWIZ-1 or a salt thereof.

10. The composition according to claim 9, characterized in that The PD-1 monoclonal antibody is selected from tislelizumab, carrelizumab, nivolumab, pembrolizumab and sintilimab; and / or The tumor is liver cancer.

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